Replication interruptions at the sites of DMA damage or other barriers are believed to be a primary cause of mutagenesis, genomic rearrangements, and lethality in cells. The RecF pathway is one of the two major pathways of recombinational repair essential for successful restart of replication. The RecF, -O and -R proteins, also known as recombination/replication mediators, form the core of the RecF pathway. They are essential for the loading of RecA recombinase onto DNA, the event which triggers the SOS response, and is important to maintain the stability of stalled replication forks, to resolve aberrant DNA structures, and to resume replication from the point of disruption. These proteins have functional counterparts in all organisms. The mechanism of their activity even in bacteria is poorly understood, and is a long term goal of our research. One of the important unanswered questions about RecFOR activities is what are the mechanisms of association/dissociation of RecFOR complexes and their interactions with DNA. We will address this question in our proposal by performing structural studies of RecF and RecFOR complexes, and by studying the mechanism of the initial steps of RecFOR reaction: RecF DNA binding and the recruitment of RecR by RecF to DNA. The hypotheses that the ATP-driven dimerization plays a central role in RecF activity and regulates RecF interactions with DNA and/or with RecR will be investigated in the following aims: 1) the crystal structure of RecF will be solved at high resolution to reveal specific features of the SMC domain and the novel structure of RecF specific domain;2) ATP-dependent dimerization of RecF alone and upon DNA binding will be directly measured;structure-guided mutagenesis of RecF will be utilized to further study the role of SMC-like dimerization in ATP hydrolysis and DNA binding;and 3) the assembly state of the RecF/RecR/DNA complex, the pathway of its formation, and the role of the RecF ATP-dependent dimerization will be studied with wild type and mutant proteins. We also will attempt to co-crystallize RecFOR complexes to study their interactions at the atomic resolution level. Together with our collaborative studies, in which selected mutants will be tested for their ability to facilitate RecA nucleoprotein filaments assembly in vitro and for their effect on the replication recovery in UV irradiated cells, the proposed research will provide significant and novel information on recombination/replication mediators. A new detailed mechanistic understanding of RecF interactions with DNA and RecR during the repair of stalled replication forks will be obtained thus advancing our understanding of the replication restart and recombination repair processes. It will help formulate future studies of similar processes in eukaryotes where their malfunctions are highly associated with tumorigenesis.